Maintenance of genome stability is extremely important in the cell cycle, with genomic instability being the hallmark of cancer cells. DNA re-replication is one type of genomic instability with replication origins firing multiple times during the same cell cycle. Although several genes that regulate DNA re-replication have been identified, the mechanisms behind them remain to be determined. Moreover, whether DNA re-replication control can be used for cancer therapy is unknown. The aim of this proposal is to investigate the mechanisms by which Cdk1 prevents DNA re-replication during G2/M phase in both human cancer and normal cells, to screen for small molecules that selectively cause DNA re-replication in cancer cells, and to perform genome-wide studies of DNA re-replication control. This study will be useful not only to understand one facet of how our cells maintain a stable genome, but also to provide us with useful therapeutic targets and potential compounds for the treatment of cancer. Therefore, this proposal is relevant to the mission of NIH/NCI in its pursuit of fundamental knowledge about living systems and supporting research, training, and other programs with respect to the cause, diagnosis, prevention, and treatment of cancer. The Specific Aims of this proposal are: Aim 1. Investigate the role of Cdkl in regulating Orel activity to prevent DNA re-replication during G2/M. In particular, we will investigate the role of phosphorylation of Orel by Cdk1 kinases during G2/M and whether Orel is involved in re-replication regulation during G2/M. Aim 2. Screen for small molecules by a HTS assay to identify small compounds that selectively cause DNA rereplication in cancer cells. Aim 3. Perform cell-based genome-wide studies of DNA re-replication control via a HTS assay by siRNA transfection to identify new genes that involve in DNA re-replication control. My ultimate career goal is to become an independent researcher in the field of cancer research. To accomplish this, currently I am investigating the mechanisms that cause genome instability in cancer cells and its application for cancer therapy, resulting in the application for this award.